Therapeutic Goods Order No. 54 - Standard for Disinfectants and Sterilants (25/10/1996)
3. Test Inoculum3. Test Inoculum
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This test method has been reproduced with the kind permission of the author and publisher from an original paper published in the “Australian Journal of Hospital Pharmacy”, Vol 8, No 4; 1978 (152‑155)
1. Principle
The method, as applied to Hospital Grade Disinfectants or Sanitisers, is essentially that given by Kelsey & Maurer (1) for testing disinfectant performance. It is set out in a form suitable for attachment to a regulatory minimum standard for disinfectants and antiseptics. For wider application of the test refer to supplementary note A.
The disinfectant is tested at the dilution recommended by the manufacturer on the product label. The test consists of challenging the diluted disinfectant with bacterial inoculum, withdrawing a sample after a given time and culturing the sample in a suitable recovery medium. After this sampling, the mixture is again challenged by a second inoculum and after a second interval is again sampled for culturing. The sample is passed or failed according to the extent of growth shown in the two cultures sampled. The test may be performed with or without the addition of sterile yeast as an organic soil. (Options B and A respectively) or both, according to the use‑situations advocated on the label of the product under test.
Table 1. Selection of test parameters for classes of disinfectant and antiseptic using the TGA Disinfectant Test.
For Household Grade disinfectants, the first two organisms listed and the second challenge are omitted, while Option C (nutrient broth) is selected as the choice of simulated soil. For antiseptics, the second challenge is again omitted, while Option D (serum) is selected as the choice of soil.
2. Media
All media must be contained in capped glass containers. Where media are stored, the containers must be sealed tightly or refrigerated.
2.1 Sterile Hard Water
2.1.1 Dissolve 0.304g anhydrous calcium chloride and 0.065g anhydrous magnesium chloride in glass‑distilled water, and make up to one litre.
2.1.2 Dispense into glass containers and sterilize by autoclaving at 1210 +10 C for 15 minutes.
2.2 Yeast Suspension
2.2.1 Weigh 200g of moist compressed baker's yeast. Cream by the gradual addition of sterile hard water using a heavy glass rod for stirring. Decant the creamed portion into a flask, add more water to any lumpy residue remaining and repeat the creaming and decantation until no residue remains and 500ml of water has been used.
2.2.2 Shake the contents of the flask vigorously and strain through a 100‑mesh sieve, breaking down any remaining lumps.
2.2.3 Add 500ml sterile hard water, shake vigorously and adjust the pH to 6.9‑7.1 with 1N Sodium hydroxide.
2.2.4 Transfer 50ml, 100ml or 200ml of the yeast solution into screw‑capped bottles.
2.2.5 Autoclave at 1210 + 10C for 15 minutes and allow the autoclave to cool without releasing pressure. Store cold but not freezing.
2.2.6 Dry two Petri dishes to constant weight. Into each, pipette 25ml of sterilised yeast suspension, and dry to constant weight at 1000C. Calculate the average solids content of the suspension.
2.2.7 Before use, pipette 25ml of the sterilised yeast suspension into a beaker. Determine the pH using the glass electrode, and determine the volume of 1N sodium hydroxide solution needed to adjust the pH to within the range 6.9 to 7.1.
2.2.8 Immediately before use, add to each bottle of sterilised yeast, a volume of sterile hard water and a volume of 1N sodium hydroxide calculated to adjust the concentration of dry yeast to 5.0% and the pH to within the range 6.9‑7.1. Discard prepared yeast 3 months after preparation.
2.3 Medium for Growth of Test Organisms
2.3.1 Prepare a 10% w/v dextrose solution in distilled water, and sterilise by autoclaving at 1210 + 10C for 15 minutes. Cool to room temperature.
2.3.2 Prepare Wright and Mundy medium following the author's procedure (2) or from a commercial product of the same composition (Note B) and sterilise by autoclaving at 1210 + 10C for 15 minutes. Cool to room temperature.
2.3.3 To each litre of Wright and Mundy medium prepared in 2.3.2 add 10ml sterile dextrose solution prepared in 2.3.1.
2.3.4 Aseptically dispense in either 10ml or 15ml amounts, as preferred.
2.3.5 This medium is referred to as Wright and Mundy dextrose medium.
2.4 Recovery Medium
2.4.1 Prepare nutrient broth as follows or from a commercial product of the same composition (Note B):‑
Add the following to 970ml of water and dissolve by heating.
Beef Extract Powder 10g
Peptone 10g
Sodium Chloride 5g
Adjust the pH to 8.0‑8.4 using 1N Sodium Hydroxide.
Boil for 10 minutes and filter. Cool.
2.4.2 To each litre of nutrient broth solution prepared in 2.4.1 add 30g polysorbate 80 (Note B).
2.4.3 Adjust pH to 7.2‑7.4, using 1N Sodium hydroxide.
2.4.4 Autoclave at 1210 + 10C for 15 minutes, and immediately shake well to disperse the polysorbate 80.
2.4.5 Dispense aseptically in 10ml amounts into sterile capped glass tubes.
3. Test Inoculum
3.1 Test Organisms
The following 4 organisms are to be used, except where prescribed.
Pseudomonas aeruginosa NCTC 6749
Proteus vulgaris NCTC 4635
Escherichia coli NCTC 8196
Staphylococcus aureus NCTC 4163
3.2 Preparation of Inoculum
3.2.1 Incubate the contents of an ampoule of freeze‑dried culture overnight at 370 + 10C in Wright and Mundy dextrose medium.
3.2.2 Inoculate the incubated culture onto nutrient agar slopes in McCartney bottles. Store for up to 3 months at 40 + 10C.
3.2.3 At a suitable period before the test is to be conducted, sub‑culture from an agar slope into 10ml or 15ml quantities of Wright and Mundy dextrose medium. Incubate at 370 + 10C for 24 + 2 hours.
3.2.4 Sub‑culture from the medium in 3.2.3 into fresh medium, using an inoculating loop of 4mm in diameter. Incubate at 370 + 10C for 24 + 2 hours.
3.2.5 Repeat step 3.2.4 daily. For the test procedure use only those cultures which have been sub‑cultured at least 5, and not more than 14 times.
3.2.6 Filter test cultures of P. aeruginosa and S. aureus through sterile Whatmans No. 4 filter paper.
3.2.7 Centrifuge all test cultures until cells are compact, and remove supernatant with a Pasteur pipette.
3.2.8 Resuspend test organisms in the original volume of liquid (i.e. 10ml or 15ml), and shake for 1 minute with a few sterile glass beads.
3.2.8.1 For Option A, resuspend in sterile hard water.
3.2.8.2 For Option B, resuspend in a mixture of 4 parts yeast suspension (prepared as in 2.2) to 6 parts sterile hard water.
3.2.8.3 For Option C, resuspend in nutrient broth (prepared as in 2.4.1 and 2.4.3 and sterilised by autoclaving).
3.2.8.4 For Option D, resuspend in sterile hard water; dilute twice 1 + 9 in sterile hard water; then add 8ml of the last dilution to 2ml sheep serum previously inactivated at 560C for 20 mins. and sterilised by filtration.
3.3 Enumeration of Inoculum
Immediately before testing, sample the resuspended inoculum and enumerate using 10‑fold dilutions in quarter‑strength Ringer's solution and the pour‑plate technique. The number subsequently counted must represent not less than 2 x 108 or more than 2 x 109 organisms per millilitre (or 1 x 108 ‑ 1 x 107 using Option D) or the test is considered invalid. Retain tube containing 10‑7 dilution for use in controls (7.3 and 7.4).
4. Disinfectant Dilutions
Quantitatively dilute a sample of the disinfectant to the specified extent, using sterile hard water as diluent. Use not less than 10ml or 10g of sample for the first dilution, and not less than 1ml of any dilution to prepare subsequent dilutions. Make all dilutions in glass containers on the day of testing. The glass containers must be twice rinsed in glass‑distilled water, and sterilised.
5. Temperature
Where air‑conditioning does not maintain test solutions at 210 + 10C, hold the containers in which the test is to be carried out in a waterbath at this temperature.
6. Test Procedure
Perform the following test using each of the four test organisms (3.1) except where the Standard directs otherwise. It is not necessary to test with all organisms simultaneously.
6.1 Add 3ml of diluted disinfectant to a capped glass container.
6.2 Start a timing device. Immediately inoculate disinfectant with 1ml of culture (prepared in 3.2) and mix by swirling.
6.3 At 8 minutes, subculture one drop (0.02ml + .002ml) into each of 5 tubes containing recovery broth. To ensure delivery of 0.02ml into the first tube of recovery broth at exactly 8 minutes, it will be necessary to withdraw a suitable amount from the disinfectant test mix shortly beforehand. This must be immediately preceded by vortexing. Surplus sample must be returned to the test mix. (See Note D).
6.4 Except where prescribed, at 10 minutes, inoculate disinfectant with a further 1ml of culture, and mix by vortexing.
6.5 Except where prescribed, at 18 minutes, proceed as in 6.3.
6.6 Mix the contents of all tubes of recovery broth by vortexing. Incubate at 370 + 10C for 48 + 2 hours.
6.7 Examine for growth and record results.
6.8 For each test organism repeat steps 6.1‑6.7 on each of 2 subsequent days, using a fresh disinfectant dilution and a freshly prepared bacterial suspension.
7. Controls
7.1 Recovery broth contamination
Incubate one uninoculated tube of recovery broth at 370 + 10C for 48 + 2 hours and examine for growth. If growth occurs, the test is considered invalid due to contamination of the recovery broth.
7.2 Disinfectant contamination
To 1 tube of recovery broth, add 0.02ml of diluted disinfectant. Incubate at 370 + 10C for 48 + 2 hours. If growth occurs, the test is considered invalid. Growth in 7.2 but not 7.1 indicates contamination of the disinfectant test solution.
7.3 Fertility Test
To 1 tube of recovery broth, add 1.0ml of the 10‑7 dilution retained in 3.3. Incubate at 370 + 10C for 48 + 2 hours and examine for growth. If no growth occurs, the test is considered invalid.
7.4 Inactivator Efficacy
To 1 tube of recovery broth, add 0.02ml of diluted disinfectant and 1.0ml of the 10‑7 dilution retained in 3.3. Incubate at 370 + 10C for 48 + 2 hours, and examine for growth. If no growth occurs, the test is considered invalid. Growth in 7.3 but not in 7.4 indicates inadequate inactivation of the disinfectant.
8. Procedure in case of invalid controls
When any control renders the test invalid, the test is to be repeated. Fresh recovery broth is to be used if growth occurred in control 7.1 or if no growth occurred in controls 7.3 or 7.4.
Should disinfectant contamination be indicated by control 7.2 on both occasions, the disinfectant is considered to fail the test. Should inadequate inactivation of the disinfectant be indicated by control 7.4 on both occasions, the test is considered invalid (Note C).
9. Results
The dilution test passes the test if there is no apparent growth in at least two out of the five recovery broths specified in 6.3 and no apparent growth in at least two of the five recovery broths specified in 6.5 on all three occasions, using all four organisms.
10. References
(1) Kelsey, J.C. and Maurer Isobel, M. Pharmaceutical Journal (UK) 213: 528‑530, (1974).
(2) Wright Eleanore, S. and Mundy, R.A. Journal of Bacteriology 80: 279‑280, (1960).
11. Supplementary Notes
A. For investigational, developmental or comparative purposes, it will be useful to add a third challenge thus performing a true capacity test, and to test at dilutions above and below the prescribed dilution. In such cases, Kelsey & Maurer's recommendations regarding the timing and organisation of the test should be carefully consulted. Abbreviations of the test may be considered for the routine test of production batches.
B. Wright & Mundy medium is commercially available as “Bacto Synthetic Broth”, A.O.A.C. Code No. 0352 (Difco Ltd.). The nutrient broth to be used is available as “Nutrient Broth ‑ No. 2” (Oxoid Ltd.).
C. Where inadequate inactivation is indicated, investigations should be conducted to find an effective inactivator. Refer Mackinnon, I.H.J. Hyg (London) 73: 189‑195, (1974).
D. The Oxford P‑7000 sampler system with disposable plastic tips is recommended for the withdrawal of samples for subculturing.